GRAM NEGATIVE COCCOID BACTERIUM

Description

Technical Field

[0001] The present invention relates to an isolated bacterium and uses of the bacterium to diagnose gastrointestinal diseases caused by the bacterium.

Background Art

[0002] Gastrointestinal disease is a common affliction in animals and humans. Although many aetiological agents including viruses, bacteria and protozoa, have been recognised to cause this disease, there are still many clinical cases from which no causative agent can be identified. Furthermore, many cases do not respond to standard medical treatment regimes suggesting that some unknown agent or agents may be involved.

[0003] There have been several new microorganisms identified recently that have been shown to cause gastrointestinal disease in animals including humans. In 1984, Marshall and Warren (Lancet 1984: 1312-1314) described Campylobacter-like, spiral, Gram negative bacteria 2.5 µm in length and 0.5 µm in diameter in patients with gastritis and peptic ulcers, now known as Helicobacter pylori. A urea broth-based test to detect Helicobacter pylori presence in upper gastrointestinal biopsies is described by Weiss et al in Diagn Microbiol Infect Dis 1996;24:61-64. In 1987, Dent et al (Lancet 1987 ii:96) reported a new Gram negative spiral bacterium. 3.5- 7.5 µm in length and 0.9 µm in diameter in patients with gastritis and named it as Gastrospirillum hominis. This organism is now called Helicobacter heilmanii.

[0004] The present inventor has isolated and identified a new bacterium that is implicated in gastrointestinal disease in humans.

Disclosure of Invention

[0005] A new coccoid form of bacterium, distinct from the coccoid form of Helicobacter pylori, which can colonise the human stomach was identified and isolated from a patient with dyspeptic symptoms and found to suffer from a diffused gastritis associated with an erosive gastroduodenitis and multiple superficial ulcerations- The bacterium was resistant to usual triple antibiotic therapy composed of tetracycline, metronidazole, and an H₂ antagonist. Symptomatic remission and histological and electron microscopic resolution of the gastro-duodenal pathology following a proton pump inhibitor suggests possible role in the pathogenesis of erosive gastroduodenitis and multiple superficial ulcerations. The organism is coccoid form and varying in size 0.2 - 0.75 µm in diameter, most being 0. 3 - 0.5 µm in diameter, with outer membrane pili. The bacterium has been named by the present inventors as Okadaella gastrococcus Gen. nov , Sp. nov.

[0006] In a first aspect, the present invention consists in an isolated Okadaella gastrococcus bacterium capable of causing gastrointestinal disease in an animal, wherein said Okadaella gastrococcus bacterium has the characteristics of the Okadaella gastrococcus deposited under AGAL Accession Number NM 98/08610.

[0007] Preferably, the bacterium has the following characteristics:

Gram negative;

coccoid;

culturable under microaerophilic and anaerobic conditions;

urease-negative under culture for 2 weeks;

catalase-negative;

oxidase-negative;

contains no flagella;

bacterial colonies do not fluoresce under ultraviolet light (360 nm);

colonies observed as numerous round "dots" in the Warthin-Starry silver stain; and

non Helicobacter pylori and non Helicobacter heilmanii.

[0008] A sample of Okadaella gastrococcus was deposited under the provisions of the Budapest Treaty with the Australian Government Analytical Laboratories (AGAL) on 22 September 1998 and given Accession Number NM 98/08610.

[0009] In a second aspect, the present invention consists in a method of diagnosing gastrointestinal disease in an animal caused by Okadaella gastrococcus, the method comprising identifying the presence of the bacterium according to the first aspect of the present invention in a clinical specimen obtained from the animal.

[0010] The bacterium may be detected directly by culturing the bacterium from a clinical specimen, identifying the bacterium by microscopy, or may be identified indirectly by the use of antibodies to the bacterium. The clinical specimen may be a biopsy, stool specimen, blood sample, or the like. It will be appreciated that the discovery of the bacterium and its association with gastrointestinal disease will allow its detection by any of the known methods of the art. It will also be appreciated that molecular detection methods like polymerase chain reaction (PCR) can also be used to identify the presence of the bacterium in a clinical specimen.

[0011] The presence of the bacterium in an animal that does not have clinical signs of gastrointestinal disease may also be indicative of pre-disposition of that animal to gastrointestinal disease. Therefore, the scope of the second aspect of the present invention also includes screening for the presence of the bacterium in animals as a means of detecting animals that may be susceptible to gastrointestinal disease caused by this bacterium.

[0012] Preferably, the animal is a human.

[0013] In a third aspect, the present invention consists in use of the bacterium according to the first aspect of the present invention to develop diagnostic tools or aids for the detection or diagnosis of disease caused by the bacterium. Such diagnostic tools or aids include molecular probes derived from the genome of the bacterium and antibodies.

[0014] Throughout this specification, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

[0015] In order that the present invention may be more clearly understood, preferred forms will be described with reference to the accompanying drawings.

Brief Description of Drawings

[0016] 

Figure 1 its a scanning electron micrograph of gastric biopsy specimen showing the coccoid bacteria in the damaged mucosal layer, with an erythrocyte and leukocyte for size comparison, x 2,640. Bar = 10 µm

Figure 2 is an enlargement of the scanning micrograph shown in Figure 1.

Figure 3 is a negative stain electron micrograph of the coccoid bacteria, x 37,500. Bar = 1 µm.

Figure 4 shows transmission electron micrographs of the bacterium according to the present invention.

Description of the Invention

[0017] In 1983, Warren and Marshall described Campylobacter-like, Gram negative, S-shaped bacilli 2.5 µm in length and 0.5 µm in diameter in patients with gastritis and peptic ulcers, now known as Helicobacter pylori. In 1987, Dent et al reported gram negative spiral bacteria, 3.5 - 7.5 µm in length and 0.9 µm in diameter in patients with gastritis and named it Gastrospirillum hominis, now called Helicobacter heilmannii.

[0018] There is no published information available regarding the identification and isolation of other microoganisms from patients with dyspepsia, even though dyspepsia is one of the most common medical complaints in the World. The present inventors have been attempting to culture, isolate and identify new microorganisms from patients with dyspepsia. A case of a young Japanese who suffers from an erosive gastro-duodenitis with multiple superficial ulcerations with unknown aetiology. The present inventors succeeded to isolate a coccoid form of organism which is not H. pylori from this patient. Chan et al reported that the presence of coccoid forms of H. pylori is the dominant feature of adenocarcinoma of the stomach. However, international conflicts exist whether the coccoid form of H. pylori is a viable dormant form or nonviable degenerative form of the curvilinear microorganism. Therefore, it is very important to distinguish the coccoid form of the microorganism from Helicobacter species which could colonise human gastrointestinal tract. The endoscopic, microbiological, histological and ultrastructural and molecular biological findings of the new micro-organism is provided below.

CASE REPORT

[0019] A 20 year old man presented with dyspepsia, indigestion, heartburn and occasional history of gastro-oesophageal reflux. He complained of pre- and post-prandial pain which was relieved by occasional "burping". He denied abdominal bloating, nausea, or vomiting. He also denied a history of melena or recent changes in his bowel habit, except that he suffered from diarrhoea 3-4 times/day around the time when he was found to have duodenitis and multiple duodenal erosion and superficial ulcerations. He denied the use of NSAIDs, his smoking habit was 3-10 cigarettes/day and drinking habit was 375 ml beer per day until this episode. Family history was negative for peptic ulcer disease and gastro-intestinal malignancy. As his condition did not respond to daily treatment with a H₂ antagonist for a month, and his H. pylori serology test (HEL-pTEST^™ II, AMRAD) was positive, he underwent a videoscope endoscopic examination (Olympus GIF XQ 230) and biopsy. As the routine procedure, at least 6 - 10 biopsy specimens were collected from gastric body and antrum for histological and microbiological examinations. The biopsy specimens and gastric aspirates were cultured under a microaerophilic and anaerobic conditions at 35° C. The biopsy specimens were tested for urease production in a Christensen's urea broth and examined under a microscope with Gram stain. Although the histological and microbiological examination did not reveal the presence of Helicobacter-like microorganisms, he was treated with two weeks course of the triple combination therapy with tetracycline 1.5 g, metronidazole 600 mg and ranitidine 300 mg in divided dose daily (causing greater than 98% successful eradication of H. pylori). Further follow-up endoscopic examinations were performed. In all occasions, the endoscopic appearance of the stomach was a generalised gastritis with numerous gastric erosions and multiple surface ulcerations. An erosive duodenitis and multiple surface ulcerations were also seen at the same time. In all occasions, specimens collected from the gastric body and antrum were submitted for histological examination in which were all showed an erosive gastritis with focal superficial ulcerations and focally increased numbers of neutrophils and chronic inflammatory cells in the lamina propria. The aetiology of the gastro-duodenopathy could not be identified by the histological examination. Some epithelial regeneration was seen only in the specimen collected at the time of the third endoscopic examination following one month treatment with omeprasole 20 mg b.d. Therefore, he was treated with omeprasole 20 mg b.d for 4 months and the fourth endoscopic examination was performed. The endoscopic appearance was hyperaemic erythematous and congested gastric body and antrum and duodenum without gastro-duodenal erosion and ulcerations. The histological examination revealed complete resolution of the erosive gastro-duodenitis and multiple superficial ulcerations. The scanning electron microscopic examination identified some-what enlarged and swollen scattered single coccoid microorganisms in some areas on the surface of gastric epithelium. There were no electron microscopically identifiable surface erosion nor ulceration in the stomach nor duodenum. Adequately regenerated microvilli were observed in the stomach and duodenum. While examining the Gram stained specimen, the present inventors noted Gram negative coccoid bacteria and succeeded to culture the microorganism. The presence of the coccoid organism in the gastric aspirates was also confirmed by the microbiological and electron microscopic examinations. The scanning and transmission electron microscopic examinations identified abundant coccoid forms of bacteria beneath the gastric mucous layer, on the gastric epithelial cells and the clusters of coccoid forms of bacteria in the erosive lesion of the gastric mucosa and in the superficial ulcers. The characteristics of the cultured organism were studied following Gram (G) staining, Haematoxylin and eosin (H&E) stain and Warthin-Starry silver (WSS) stain before subjecting to reviewing all the histological specimens. The bacteria were difficult to see in H & E stained histological sections. When they could see, the microorganism was recognised as a basophilic clustered mass. The organism was observed as a numerous round "dots" in the WSS. The bacteria were abundant in the superficial layer of the gastric mucosa, and could be identified in the erosive lesion of the gastric mucosa and gastric pits. The organism was urease-, catalase-, and oxidase-negative. They converted arginine-β-naphthylamine to arginine. The bacteria lacked flagella but were surrounded by fine microfilamentous hair like pili. The 16S rRNA sequencing study confirmed that the coccoid form of bacteria were not phylogenically related with the coccid form of H. pylori but were closer to Haemophilus haemolyticus. DNA comparison studies of the coccoid form and H. haemolyticus demonstrated that the microorganisms are different. The response to factor X and V which are required to determine the genus Haemophilus were also tested with the results being negative for both. The growth conditions and environment of Haemophilus are not suitable for the coccoid form of the organism. The growth rate was totally different from Haemophilus which growth can be detected after 24 hours incubation. The coccid organism, however, takes at least 7 days to give any hint of colony formation. The colony appearance and morphology are also different. The biochemical test results were totally different from Haemophilus and could not be identified by the commercially available identification kit, Neisseria/Haemophilus identification card (biomerieux, Vitek), nor by RapID ANA II system (Innovative Diagnostic Systems) for identification of anaerobic organism. Chocolate agar was used as the standard for culturing Haemophilus, which is not optimal for the new organism.

[0020] Immunoperoxidase stain study was performed on a paraffin block section to exclude the possibility of coccoid forms of H. pylori colonisation. There was no H.pylori sera reaction identified.

DISCUSSION

[0021] The microaerophilic and anaerobic gram-negative coccoid form of bacteria which colonised the human stomach mucosa have not been cultured previously, and their association with an erosive gastro-duodenitis and multiple superficial ulcerations have not been described. The main reason why the new bacteria could not have been detected previously is probably due to their size and appearance which resemble to cell debris such as cytoplasmic granules in particular expelled chromatins from cell nuclei on inflamed, erosive and ulcerated gastric mucosal cells. The organism may have been seen but regarded as oropharygeal contaminants, or superseded by the presence of H. pylori and regarded as commensals. The possibility also exists that the bacteria were misinterpreted and thought as the coccoid form of H. pylori. At first, it was very difficult to detect the presence of the organism in histological specimens even by very well trained and experienced pathologists until the size, nature and characteristics of the isolated organism which were stained with G, H&E and WSS was recognised. Second, it is quite easy to discard and neglect the cultured colonies as they are very small and about 0.5 -1 mm in diameter after about 1 week incubation. The Gram stained cultured specimen often appears like a Gram negative artefact rather than clear distinct microorganism as they are densely packed and clustered bacteria and appear like "sand". The other reason could have been due to the neglect of gastric microbiology and the concept that the stomach is a sterile organ as stated by Warren when he identified S-shaped bacilli in patients with gastritis.

[0022] It is important to make an accurate diagnosis of the gastro-duodenal pathology if it is associated with bacterial colonisation like H. pylori, H. heilmannii, Okadaella infection (Okada et al 1998) or other bacterial infections; candida (Kalogeropoulos et al 1988), cryptosporidium (Cersosimo et al 1992), as the treatment choice would be different. One may select triple therapy incorporating tetracycline and metronidazole which achieves a significantly higher successful eradication of H. pylori than others (Chiba et al 1992). Okadaella gastrococci were resistant to the combination treatment with ranitidine, tetracycline and metronidazole for 2 weeks. Considering the disappearance of the organism histologically and microbiologically following 4 months treatment with omeprazole coincided with histological and symptomatic improvement of this patient, the combination therapy with proton pump inhibitors could produce greater efficacy than other classes of acid-suppressing agents in the management of an erosive gastro-duodenitis and peptic ulcer disease due to Helicobacter and Okadaella infection.

[0023] The organism isolated by the present inventors does not fit into known genus Haemophilus, judging from the colony, cell morphology, growth characteristics, antibiotic susceptibility, biochemical tests, and DNA analysis although the new organism has close phylogenic relationships with H. haemolyticits. The genus called. Okadaella Gen. nov. (Oka.da.-ella: Jap. n. Okada; N. L. masc. n. Okadaella, named after Dr T Okada who identified the bacterium), gastrococcus Sp. nov. (Gr. n. gastr: pertaining to the stomach; Gr. n. coccus: grain) is proposed for the present microorganism. Antibodies raised against this organism and performance of immuno-histo-fluorescence and indirect immuno-histochemical studies will be useful to show that the bacteria isolated are identical to the organisms on the surface of the gastric mucosal epithelium.

Biochemical characteristics of Okadaella gastroccocus

[0024] 

Growth medium - Horse blood agar (eg. Columbia agar)

Optimal growth condition: 35 - 37°C

Growth condition: Microaerophilic and anaerobic
(CO₂: 10%, O₂: 4.86%, N₂: 85.14%)

Colony size to reach 0.5 - 1.0mm: 7 -14 days

Colony morphology: Convex, smooth, translucent

Cell body diameter: 0.3 - 0.5 µm

Cell body length: 0.2 - 0.75 µm

Mole % G + C: not determined

X factor requirement: No

Y factor requirement: No

Catalase: negative

Oxidase: negative

Urease: negative

Indole: negative

Ornithine decarboxylase: negative

Haemolysis: negative

Alkaline-phosphatase: not determined

Glucose: negative

Acid product from

L-arabinose: negative

D-galactose: negative

Maltose: negative

Sucrose: negative

Galactosidase (ONPG): negative

(ONPG: o-nitrophenyl-D-galactosidase)

Frucosidase: negative

Glucosidase: negative

Arginine dihydrolase/hydrolase: Positive

Nitrate reduction: negative

Nitrite reduction: negative

H₂S production: not determined

G-Glutamyl transpeptidase: not determined

Storage at -70°C with horse red blood cells or horse serum.

(Positive: 90% or more of strains are positive)

(Negative: 10% or less of strains are positive)

REFERENCES

[0025] 

Noach L. A., Rolf T. M., Mytgat G N. Electron microscopic study of association between Helicobacter pylori and gastric and duodenal mucosa, J Clin Pathol 1994; 47: 699-704

Ogata T. and Araki K. Electron microscopic study of the morphological changes of gastric mucous cell induced by Helicobacter pylori in human gastric ulcers, J. Submicrosc PatholT. 28 (2) 255-264, 1996.

Bode G., Malfertheiner F. andDitschuneit H. Pathogenic implications of ultrastructural findings in Campylobacter pylori related gastroduodenal disease Scand J Gastroenterol 1998, 23 (suppl 142), 25-39.

Okada T., Fong, J., Adkins, G., Tskileas G., Bradley, J. Dyspepsia associated with bacterial colonization: a new bacterium isolated from the human stomach. Hong Kong Med. J.1998, 4(Suppl), 28

Cersosimo, E., Wolkowske, C.J., Rosenblatt, J.E., Ludwig, J. Isloated antral narrowing associated with gastrointestinal cryptosporidosis in acquired autoimmune deficiency syndrome. Mayo Clin, Proc., 1992, 67, 553-556.

Chiba, N., Rademaker, J.W., Rao, B.V., Hunt, R.H. Eradication of Helicobacter pylori- meta-analysis to determine optimal therapy. Gut, 1991, 32, A1220.

Kalogeropoulos, N.K., Whitehead, R. Campylobacter-like organism and candida in peptic ulcer and similar lesion of the upper gastrointestinal tract: a study of 247 cases. J. Clin. Path., 1988, Vol 41, No 10, 1093-1098.

Biochemical characteristics of Okadaella (O) and Haemophilus (H) organisms

[0026] Gc, gastrococcus; Gf, gastrofilamenti; inf, influenzae; par, parainfluenzae; haem, haemolyticus; HP, Helicobacter pylori

Characteristics	OGc	OGf	H. inf	H. par	H. haem	HP
G. medium	H.B.A.	H.B.A.	C.A.	C.A.	C.A.	H.B.A.
Optimal G. temp	35-37	35-37	35-37	35-37	35-37	35-37
G condition	MA&An	MA&An	A/FAn	A/FAn	A/FAn	MA&An
Colony size to reach 0.5-1.0 mm	7-14 days	7-14 days	24hrs	24hrs	24hrs	3-4 days
Colony morph.	convex	convex	convex	convex	convex	Convex
	TL	TL	G/TL	G/Y	TL	TL
	smooth	smooth	smooth	smooth	smooth	Smooth
Cell diameter µm	0.3-0.5	0.5	0.3-0.5			0.5
Cell length µm	0.2-0.75	>20	0.5-3.0			2.5-4.0
Mole% G + C (Tm)	?	?	37-44	40.41	39	35-44
Xfactor requirement	No	(No0	Yes	Yes	Yes	No
Vfactor requirement	No	No	Yes	No	Yes	No
Catalase	-	(-)	+	+/d	+	+
Oxidase	-	(-)	+	+	+	+
Urease	-	(-)	+/-	+/-	+	+
Indole	-	(-)	+/-	-	d	-
Ornithine decarboxy	-	(-)	+/-	+/-	-	?
Hemolysis	-	(-)	-	-	+	-
Alkaline phosphatase	?	?	+	+	+	+
AMP reaction
Glucose	-	(-)	+	+	+	?
Acid production from
L-Arabinose	-	(-)	-	-	-	?
D-Galactose	-	(-)	+	+	+
Maltose	-	(-)	+	+	+
Sucrose	-	(-)	-	+	-
-Galactosidase (ONPG)	-	(-)	-	+/d	-
-Fucosidase	-	(-)	-	-	-
-Glucosidase	-	(-)	-?	-	-
-Glucosidase	-	(-)	-	-	-
Arginine hydrolase	+	(+)	-	-	-
Nitrate reduction	-	(-)	+	+	-	-
Nitrate reduction	-	(-)	-	+	d
H2S production	?	?	-	+	+	-

+, 90% or more of strains are positive; d, 11-89% of strains are positive. ONPG, o-nitrophenyl- -Dgalactoside. H.B.A., horse blood agar; CA, chocolate agar. MA&An, microaerophilic and anaerobic condition; A/FAn, aerobic/facultatively anaerobic.

Claims

1. An isolated Okadaella gastrococcus bacterium wherein said Okadaella gastrococcus bacterium has the following characteristics of the Okadaella gastrococcus deposited under AGAL Accession Number NM 98/08610:

capable of colonizing the human stomach;

capable of causing gastrointestinal disease;

coccoid;

0.2-0.75 µm in diameter and preferably 0.3-0.5 µm in diameter;

has outer membrane pili; and

resistant to triple antibiotic therapy comprising tetracycline, metronidazole and an H₂ antagonist.

2. The isolated Okadaella gastrococcus bacterium according to claim 1 and having the further following characteristics:

Gram negative;

culturable under microaerophilic and anaerobic conditions;

urease-negative under culture for 2 weeks;

catalase-negative;

oxidase-negative;

contains no flagella;

bacterial colonies do not fluoresce under ultraviolet light (360 nm); and

colonies observed as numerous round "dots" in the Warthin-Starry silver stain.

3. The isolated Okadaella gastrococcus bacterium according to claim 1 or 2 comprising the Okadaella gastrococcus deposited under AGAL-Accession Number NM 98/08610.

4. A method of diagnosing gastrointestinal disease in an animal caused by the bacterium Okadaella gastrococcus, the method comprising detecting the presence of the bacterium as defined in any one of claims 1 to 3 in a clinical specimen obtained from the animal.

5. The method of claim 4 wherein the clinical specimen comprises a gastric aspirate.

6. The method of claim 5 wherein the gastric aspirate comprises gastric mucosa.

7. The method of claim 5 wherein the gastric aspirate comprises superficial ulcers.

8. The method of claim 5 wherein the gastric aspirate comprises gastric epithelial cells.

9. The method according to claim 4 wherein the clinical specimen is a biopsy, stool specimen, or blood sample.

10. The method according to any one of claims 4 to 9 wherein detecting the presence of the bacterium in a clinical specimen comprises identifying the bacterium by microscopy.

11. The method according to any one of claims 4 to 9 wherein detecting the presence of the bacterium in a clinical specimen comprises using antibodies to the bacterium.

12. The method according to any one of claim 4 to 9 wherein detecting the presence of the bacterium in a clinical specimen comprises identifying the bacterium by polymerase chain reaction (PCR) amplification of a part of the bacterium genome comprising 16S rRNA.

13. The method according to any one of claims 4 to 9 wherein detecting the presence of the bacterium in a clinical specimen comprises culturing the bacterium from the a clinical specimen.

14. The method according to claim 13 further comprising identifying the bacterium by microscopy.

15. The method according to claim 13 further comprising identifying the bacterium using antibodies to the bacterium.

16. The method according to claim 13 further comprising identifying the bacterium by polymerase chain reaction (PCR) amplification of a part of the bacterium genome comprising 16S rRNA.

17. The method according to any one of claims 4 to 16 wherein the clinical specimen is from a human.

18. Use of the isolated a bacterium according to any one of claims 1 to 3 in the development of a diagnostic tool or aid for the detection or diagnosis of disease caused by the bacterium.

Ansprüche

1. Ein isoliertes Okadaella gastrococcus-Bakterium, wobei das Okadaella gastrococcus-Bakterium die folgenden Charakteristika des unter der AGAL-Zugangsnummer NM 98/08610 hinterlegten Okadaella gastrococcus aufweist:

in der Lage, im Magen des Menschen Kolonien zu bilden;

in der Lage, eine gastrointestinale Erkrankung zu verursachen; kokkenähnlich;

0,2 bis 0,75 µm im Durchmesser und bevorzugt 0,3 bis 0,5 µm im Durchmesser;

mit Pili an der äußeren Membran; und

resistent gegen eine Dreifachantibiotikatherapie, welche Tetracyclin, Metronidazol und einen H₂-Antagonisten umfasst.

2. Das isolierte Okadaella gastrococcus-Bakterium gemäß Anspruch 1, welches die folgenden weiteren Charakteristika aufweist:

Gram-negativ;

in der Lage, unter mikroaerophilen und anaeroben Bedingungen kultiviert zu werden;

Katalase-negativ;

Oxidase-negativ;

enthält keine Flagellen;

Bakterienkolonien fluoreszieren nicht unter ultraviolettem Licht (360 nm); und Kolonien werden als zahlreiche runde "Punkte" bei der Warthin-Starry-Silberanfärbung beobachtet.

3. Das isolierte Okadaella gastrococcus-Bakterium gemäß Anspruch 1 oder 2, umfassend das unter der AGAL-Zugangsnummer NM 98/08610 hinterlegte Okadaella gastrococcus.

4. Ein Diagnoseverfahren für eine gastrointestinale Erkrankung bei einem Tier, welche durch das Bakterium Okadaella gastrococcus verursacht wird, wobei das Verfahren das Nachweisen des Vorhandenseins des Bakteriums wie in einem der Ansprüche 1 bis 3 definiert in einem von dem Tier erhaltenen klinischen Prüfmaterial umfasst.

5. Das Verfahren nach Anspruch 4, wobei das klinische Prüfmaterial ein Magenaspirat umfasst.

6. Das Verfahren nach Anspruch 5, wobei das Magenaspirat Magenschleimhaut umfasst.

7. Das Verfahren nach Anspruch 5, wobei das Magenaspirat Oberflächengeschwüre umfasst.

8. Das Verfahren nach Anspruch 5, wobei das Magenaspirat Magenepithelzellen umfasst.

9. Das Verfahren gemäß Anspruch 4, wobei das klinische Prüfmaterial eine Biopsie, ein Stuhlprüfmaterial oder eine Blutprobe ist.

10. Das Verfahren gemäß einem der Ansprüche 4 bis 9, wobei das Nachweisen des Vorhandenseins des Bakteriums in einem klinischen Prüfmaterial das Identifizieren des Bakteriums durch Mikroskopie umfasst.

11. Das Verfahren gemäß einem der Ansprüche 4 bis 9, wobei das Nachweisen des Vorhandenseins des Bakteriums in einem klinischen Prüfmaterial die Verwendung von Antikörpern für das Bakterium umfasst.

12. Das Verfahren gemäß einem der Ansprüche 4 bis 9, wobei das Nachweisen des Vorhandenseins des Bakteriums in einem klinischen Prüfmaterial das Identifizieren des Bakteriums durch Polymerasekettenreaktion (PCR)-Amplifizierung eines Teils des Bakteriengenoms, welches 16S rRNA umfasst, umfasst.

13. Das Verfahren gemäß einem der Ansprüche 4 bis 9, wobei das Nachweisen des Vorhandenseins des Bakteriums in einem klinischen Prüfmaterial das Kultivieren des Bakteriums von dem klinischen Prüfmaterial umfasst.

14. Das Verfahren gemäß Anspruch 13, ferner umfassend das Identifizieren des Bakteriums durch Mikroskopie.

15. Das Verfahren gemäß Anspruch 13, ferner umfassend das Identifizieren des Bakteriums unter Verwendung von Antikörpern für das Bakterium.

16. Das Verfahren gemäß Anspruch 13, ferner umfassend das Identifizieren des Bakteriums durch Polymerasekettenreaktion (PCR)-Amplifizierung eines Teils des Bakteriengenoms, welches 16S rRNA umfasst.

17. Das Verfahren gemäß einem der Ansprüche 4 bis 16, wobei das klinische Prüfmaterial von einem Menschen stammt.

18. Verwendung des isolierten Bakteriums gemäß einem der Ansprüche 1 bis 3 bei der Entwicklung eines diagnostischen Werkzeugs oder Hilfsmittels zum Nachweis oder zur Diagnose einer Erkrankung, welche durch das Bakterium verursacht wird.

Revendications

1. Bactérie Okadaella gastrococcus isolée, ladite bactérie Okadaella gastrococcus ayant les caractéristiques suivantes de l'Okadaella gastrococcus déposée sous le numéro de dépôt AGAL NM 98/08610 :

capacité à coloniser l'estomac humain ;

capacité à provoquer une maladie gastrointestinale ; cocciforme ;

0,2 à 0,75 µm de diamètre et de préférence 0,3 à 0,5 µm de diamètre ;

possède des pili au niveau de la membrane extérieure ; et

résistante à une thérapie antibiotique triple comprenant l'utilisation de tétracycline, de métronidazole et d'un antagoniste H2.

2. Bactérie Okadaella gastrococcus isolée suivant la revendication 1 et ayant les caractéristiques supplémentaires suivantes :

Gram-négative ;

cultivable dans des conditions microaérophiles et anaérobies ;

uréase-négative en culture pendant 2 semaines ;

catalase-négative ;

oxydase-négative ;

ne contient aucun flagelle ;

non-fluorescence des colonies bactériennes en lumière ultraviolette (360 nm) ; et

colonies observées sous forme de nombreux "points" arrondis dans le colorant à l'argent de Warthin-Starry.

3. Bactérie Okadaella gastrococcus isolée suivant la revendication 1 ou 2, comprenant l'Okadaella gastrococcus déposée sous le numéro de dépôt AGAL NM 98/08610.

4. Méthode pour le diagnostic d'une maladie gastrointestinale chez un animal provoquée par la bactérie Okadaella gastrococcus, méthode comprenant la détection de la présence de la bactérie telle que définie dans l'une quelconque des revendications 1 à 3 dans un échantillon clinique obtenu à partir de l'animal.

5. Méthode suivant la revendication 4, dans laquelle l'échantillon clinique comprend un produit d'aspiration gastrique.

6. Méthode suivant la revendication 5, dans laquelle le produit d'aspiration gastrique comprend de la muqueuse gastrique.

7. Méthode suivant la revendication 5, dans laquelle le produit d'aspiration gastrique comprend des ulcères superficiels.

8. Méthode suivant la revendication 5, dans laquelle le produit d'aspiration gastrique comprend des cellules épithéliales gastriques.

9. Méthode suivant la revendication 4, dans laquelle l'échantillon clinique est un échantillon de biopsie, un échantillon de selles ou un échantillon de sang.

10. Méthode suivant l'une quelconque des revendications 4 à 9, dans laquelle la détection de la présence de la bactérie dans un échantillon clinique comprend l'identification de la bactérie par microscopie.

11. Méthode suivant l'une quelconque des revendications 4 à 9, dans laquelle la détection de la présence de la bactérie dans un échantillon clinique comprend l'utilisation d'anticorps contre la bactérie.

12. Méthode suivant l'une quelconque des revendications 4 à 9, dans laquelle la détection de la présence de la bactérie dans un échantillon clinique comprend l'identification de la bactérie par amplification par réaction en chaîne avec une polymérase (PCR) d'une partie du génome de la bactérie comprenant de l'ARNr 16S.

13. Méthode suivant l'une quelconque des revendications 4 à 9, dans laquelle la détection de la présence de la bactérie dans un échantillon clinique comprend la culture de la bactérie à partir de l'échantillon clinique.

14. Méthode suivant la revendication 13, comprenant en outre l'identification de la bactérie par microscopie.

15. Méthode suivant la revendication 13, comprenant en outre l'identification de la bactérie en utilisant des anticorps contre la bactérie.

16. Méthode suivant la revendication 13, comprenant en outre l'identification de la bactérie par amplification par réaction en chaîne avec une polymérase (PCR) d'une partie du génome de la bactérie comprenant de l'ARNr 16S.

17. Méthode suivant l'une quelconque des revendications 4 à 16, dans laquelle l'échantillon clinique provient d'un être humain.

18. Utilisation de la bactérie isolée suivant l'une quelconque des revendications 1 à 3 dans l'élaboration d'un outil ou auxiliaire de diagnostic pour la détection ou le diagnostic d'une maladie provoquée par la bactérie.

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